Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/513—Organic macromolecular compounds; Dendrimers
  • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1605—Excipients; Inactive ingredients
  • A61K9/1629—Organic macromolecular compounds
  • A61K9/1635—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1605—Excipients; Inactive ingredients
  • A61K9/1629—Organic macromolecular compounds
  • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5192—Processes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
  • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
  • C08B37/0021—Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers

Definitions

• This invention generally relates to the field of cross-linked hydrogel polymers formed into microgels for use in delivery of bioactive materials such as antigens, DNA and other therapeutics.
• CTL cytotoxic T lymphocytes
• APCs antigen presenting cells
• Microparticles 0.2-5 m in diameter, have recently gained interest as delivery vehicles for protein-based vaccines because of their ability to enhance the Class I antigen presentation of protein antigens (Oh, Yu-Kyoung, Harding, C. V.; Swanson, J. A.; Vaccine. 1997, (15), 511-518; Andrianov, et al., U.S. Pat. No. 5,529,777; and Staas, et al., U.S. Pat. No. 6,321,731). Two mechanisms have been proposed to explain the ability of microparticles to enhance the Class I antigen presentation of protein antigens.
• Protein therapeutics have tremendous clinical potential and are currently being investigated for the treatment of cancer, vaccine development and for manipulating the host response to implanted biomaterials.
• the effective utilization of protein therapeutics requires the development of materials that can deliver bioactive material to diseased tissues and cells.
• the majority of protein delivery vehicles are based on hydrophobic polymers, such as poly(lactide-co-glycolide) (PLGA). See O'Hagan, D. et al., in U.S. Patent Nos. 6,306,405 and 6,086,901, and in Adv. Drug Delivery Rev, 32, 225 (1998).
• PLGA based delivery vehicles have been problematic because of their poor water solubility.
• Proteins are encapsulated into PLGA based materials through an emulsion procedure that exposes them to organic solvents, high shear stress and/or ultrasonic cavitation. This procedure frequently causes protein denaturation and inactivation as shown by Xing D et al., Vaccine, 14, 205-213 (1996). Hydrogels and microgels have therefore been proposed as an alternative protein delivery vehicle because they can encapsulate the protein in a totally aqueous environment, under mild conditions. See Park, K. et al., Biodegradable Hydrogels or Drug Delivery; Technomic Publishing Co, Lancaster, PA (1993); Peppas. N. A. Hydrogels in Medicine and Pharmacy; CRC press: Vol ⁇ , Boca Raton, FL, 1986; and Lee, K.
• a key problem in the field of hydrogel research is the development of materials that can release their contents in response to pathological stimuli, allowing for the targeting of protein therapeutics to diseased tissues and cells.
• a particularly important pathological stimulus is mildly acidic pH.
• tumors exist at acidic pHs between 6.4-6.8, and the phagolysosomes of phagocytic cells are at pHs between 4.5-5.0.
• the acidic nature of these compartments has stimulated a need for the development of hydrogels and microgels that can selectively release their contents under mildly acidic conditions.
• micron sized protein loaded hydrogels have been investigated for this purpose because they are small enough to be phagocytosed.
• micron sized hydrogels have been synthesized using crosslinkers that do not degrade under biological conditions, and hence have had limited success in drug delivery applications.
• hydrogels are synthesized using crosslinkers that contain either, amide, ester, or carbonate linkages.
• Sawhney, A. et al., Macromolecules, 26, 581-587 (1993) describe bioerodible hydrogels based on photopolymerized poly(ethyleneglycol)-co-poly( ⁇ - hydroxy acid) diacrylate macromers which utilize an ester linkage.
• Sheppard, R.C. et al., in U.S. Patent No. 5,191,015 describe an insoluble polymer with contiguous cleavable crosslinkers and functional groups, wherein the crosslinking agent is an acid degradable ketal crosslinker.
• Sanxia Sanxia.
• the present invention is directed to microgels for application in the delivery of proteins, vaccines, drugs (such as the anticancer drugs cisplatin, paclitaxel or taxitere), and other bioactive materials.
• the microgels comprise crosslinked polymer hydrogels of microparticle size, that contain bioactive materials.
• the hydrogel compositions are made using an inverse microemulsion technique (aqueous droplets in an oil or aliphatic phase) that results in microgels of a predetermined size, typically 0.1 -10 microns in diameter. A size range between 200 nm and 500 nm is optimal for phagocytosis by immune cells.
• the microgels of the current invention release their contents in response to the mild acidic conditions found in lysosomes, tumors, inflammatory tissues and the phagolysosomes of antigen presenting cells.
• the present crosslinkers will hydrolyze at a preferred pH range of 4.5 to 6.8, more preferably pH 5 to 6. Prefereably, they will completely hydrolyze within 24 hours at pH 5.
• the current invention also specifically describes hydrogels and microgels synthesized with a bisacryloyl acetal crosslinker that hydrolyzes under acidic conditions, such as in the phagolysosome, and releases the encapsulated contents of the hydrogel or microgel after entering a cell.
• the present invention provides a method of preparing a microgel composition for delivering a bioactive material to a cell, comprising the steps of (a) preparing a mixture which contains the bioactive material, a polymerizable group (i.e. the monomer to be polymerized), and a crosslinking group in an inverse emulsion where the aqueous phase contains the polymerizable group and the cross linking agent, and the aliphatic phase contains the bioactive material; (b) sonicating the mixture to achieve a pre-determined particle size, generally 0.1 to 100 Angstroms, as determined by the time of sonication; (c) polymerizing the polymerizable group and the crosslinker in the presence of the bioactive material; and (d) recovering the resulting microgel preparation having bioactive material bound inside. Polymerization is carried out according to known reaction parameters, i.e. with a known initiator (such as potassium peroxodisulfate), and an optional catalyst such as TEMED.
• a known initiator such
• the present invention thus provides an acid hydrolyzable microgel composition for delivering a bioactive molecule, comprising: an acid hydrolyzable microgel composition for delivering a bioactive molecule, comprising: a polymer backbone, which may be acrylic, dextran or other crosslinkable polymer, linked by a crosslinker; the crosslinker having the formula R 2 CH(OR 1 ) 2 , wherein R 1 is a crosslinkable, acid hydrolyzable linkage selected from one of compounds (a) -(f) of Fig.
• R 2 is Ar-X where X is a water solubilizing group selected from hydrogen, methoxy, -O-(CH 2 -CH 2 -O) n -CH 3 wherein n is from 1 to 10, -O- CH 2 -CH 2 -O-C(O)-O-Ph-NO 2 and -O-CH 2 -CH 2 -CH 2 -NH-CO-(dextran polysaccharide), said dextran polysaccharide having a molecular weight from 300 to 100,000 daltons, preferably 300-10,000 daltons; and Ar is a homocyclic aromatic radical, whether or not fused, having 6 to 12 carbon atoms optionally substituted with one to three substituents; a particle size between 0.1-10 microns and cross linkages between 1 and 20 mole percent, sufficient to physically trap the bioactive molecule within the microgel.
• X is a water solubilizing group selected from hydrogen, methoxy, -O-(CH 2 -CH 2 -
• dextran may serve as a water solubilizing group (part of the cross linker) and the polymer backbone itself.
• the crosslinker of the formula R 2 CH(OR 1 ) z containing dextran is reacted with itself in the presence of a radical source (and the bioactive material) to form the microgel composition.
• R 2 is Ar-X where X is an alkyl dextran, wherein said dextran has a molecular weight from 300 to 100,000 daltons, preferably 300-10,000 daltons and an alkyl group links the aryl group to the linked dextran.
• the R 1 groups crosslink to other R 1 groups.
• the crosslinker acts through side groups (e.g. carboxyl) on the polymer backbone formed by the polymerizing units.
• the particle prepared as described above will have cross linkages between 1 and 20 mole percent, based on the ratio of cross linker added. This degree of crosslinking is sufficient to physically trap the bioactive molecule within the microgel.
• dextran is used, the present synthetic strategy involves the preparation of an "activated dextran,” (e.g. compound 838 in Fig. 8B and compound 914 in Fig. 9B) which is attached to a crosslinker precursor through the R 1 group as represented in Fig. 2.
• the "activated dextran” has a fraction of the glucose moieties (approximately one in six) modified at the 4 ring position to carry a reactive group such as an amine group or a nitrate group for coupling to the crosslinker precursor R 1 group.
• compositions preferably contain a bioactive material which is selected from the group consisting of consisting of polysaccharides, proteins, DNA and RNA.
• the DNA may be unmethylated (e.g. bacterial plasmid) DNA, which evokes an immune response in mammals.
• the biological material may also be a protein or other chemical antigen that is delivered to the lysosome of an immune cell for antigen presentation. This provides an effective vaccine.
• composition may also be designed so that R 1 is (a) or (b) in Fig. 2 and R 2 is such that Ar is phenyl and X is methoxy.
• This composition may further comprise a bisacryloyl acetal crosslinker for use in crosslinking acrylic polymers.
• a bisacryloyl acetal crosslinker will have the formula: R 2 CH(OR ! ) 2 , wherein R 1 is selected from one of compound (a) -(d) of Fig.
• R 2 is Ar-X where X is -O-(CH 2 -CH 2 -O) n -CH 3 wherein n is from 1 to 10, and aryl is a homocyclic aromatic radical, whether or not fused, having 6 to 12 carbon atoms optionally substituted with one to three substituents.
• the present invention also comprises methods of preparing the compounds described herein.
• the methods may be particularly adapted to the synthesis of the triglyme or tetraglyme crosslinkers shown in Figs. 6 and 7 respectively. These methods comprise the steps of (a) preparing a 1-chloro oxyalkane according to the desired n; (b) reacting the compound of step (a) with with hydroxybenzaldehyde to produce an oxyalkane- benzaldehyde; (c) converting the compound of step (c) to an acetyl with a 2,2,2- trifluoracetamide; and (d) cleaving said 2,2,2-trifluoro groups and reacting the intermediate with acryloyl chloride.
• Figure 1 is a schematic diagram showing the application of the gel materials in microparticle form being applied to an antigen presenting cell.
• Figure 2 is a table showing various compositions for the present cross linkers using the generic formula R 2 -CH(-OR 1 ) 2 .
• Figure 3 is a synthetic scheme showing the manufacture of a microgel having a polymer, a cross linker, and a bioactive material, and the subsequent dissolution of the particle.
• Figure 4 is a schematic showing the synthesis of bisacrylamide methoxybenzaldehyde acetal crosslinker.
• Figure 5 is a schematic showing the synthesis of autocatalytic bisacryloyl acetal crosslinkers. Synthetic routes (5 A) to these crosslinkers and their degradation under acidic conditions (5B) are shown.
• Figure 6 is a schematic showing the synthesis of bisacrylamide triethylene glycol (triglyme) acetal crosslinker.
• Figure 7 is a schematic showing the synthesis of bisacrylamide tetraethylene glycol (tetraglyme) acetal crosslinker.
• Figure 8 is a schematic showing the synthesis of a bisacrylamide nitrochloroformate acetal crosslinker 836 and dextran microgels.
• Fig.8A shows the synthesis of the bisacrylamide nitrochloroformate acetal crosslinker 836.
• Fig. 8B shows the conjugation of the crosslinker 836 with activated dextran to yield a dextran acetal crosslinker 840.
• Fig.8C shows synthesis of dextran microgels using crosslinker 840.
• Figure 9 is a schematic showing the synthesis of bisacrylamide dextran acetal crosslinker 920 and dextran microgels.
• Fig. 9A shows the synthesis of the intermediate compound bisacrylamide amine acetal 910.
• Fig. 9B shows the conjugation of the amine acetal 910 with activated dextran to yield a bistrifluoroacetamide dextran acetal 916.
• Fig. 9C shows the modification of the intermediate acetal 916 to make the bisacrylamide dextran acetal crosslinker 920.
• Fig. 9D shows synthesis of dextran microgels using crosslinker 920.
• Figure 10 is a schematic showing dextran microgels upon acid hydrolysis and biodegredation.
• Figure 11 is a schematic showing the synthesis to activate mannose to incorporate immunostimulatory groups such as mannose into microgels to increase CTL activation.
• hydrogel herein refers to a three dimensional macromolecular network that swells in water that is formed from a cross-linked polymer.
• microgel refers to a three-dimensional hydrogel particle that is 0.1 - 10 ⁇ m in diameter.
• bioactive particle refers to a composition having a physiological effect in a cell, particularly a protein antigen, DNA, and enzyme or other organic molecule.
• acetal herein refers to a diether in which both ether oxygens are bound to the same carbon.
• crosslinker refers to a molecule with two or more functional groups that can form a three-dimensional network when reacted with the appropriate co- monomers.
• polymerizable group herein refers to monomers which polymerize upon introduction of an initiator or radical source.
• aryl herein refers to a homocyclic aromatic radical, whether or not fused, having 6 to 12 carbon atoms optionally substituted with one to three substituents, wherein said substituents are preferably N or S.
• inverse emulsion refers to an emulsion having an aqueous phase and an oil phase, wherein the continuous phase is the oil phase and the water-soluble droplets are dispersed in a continuous phase of oil.
• acrylic polymer herein refers to a polymer made from polymerizing units (monomers) that yield a polymer having a cross linkable side chain, represented as follows:
• COOH wherein said monomers are acrylic acid, acrylamide, or various monomers and mixtures thereof having hydrogen substitutions such as NH 2 at the CH group.
• loading efficiency refers to the percentage of the starting amount of bioactive material that is encapsulated per milligram of the microgels ( ⁇ g material/mg microgel) on average, based on the starting bioactive material/monomers ratio.
• alkyl-dextran refers to a dextran polymer having a glycosidic linkage from a dextran through an alkyl spacer group wherein C is less than 100 and optionally substituted with amide bonds and polyethylene glycol (-O-(CH 2 -CH 2 -O) n - where n is less than 10.
• lower alkane herein refers to an aliphatic linear or branched chain or cyclic compound of the formula C Trust-H 2n+2 , where n is between 2 and 20, such as hexane, octane, nonane or the like.
• mole percent herein refers, when used in connection with degree of crosslinking, that degree of cross linking as measured by the moles of crosslinker divided by the total moles of crosslinker and polymerizable groups.
• Fig.l shows a schematic diagram illustrating the overall composition and use of the present microgels.
• the microgels 10 of the current invention are loaded with bioactive material 40 including but not limited to, antigens, proteins, polynucleotides, polypeptides, and other bioactive material 50.
• the microgels 10 of the current invention should be synthesized with polymerizable groups 30 and a bisacryloyl acetal crosslinker 20 that hydrolyzes under acidic conditions and releases the encapsulated contents 40 in response to mildly acidic conditions.
• the mild acidic conditions found in the body such as in tumors, inflammatory tissues and in cellular compartments such as lysosomes and phagolysosomes 50 of antigen presenting cells 60 should cause the acetal group of the bisacryloyl acetal crosslinker 20 to be hydrolysed thereby degrading the microgel and releasing its contents.
• the microgels are delivered to antigen presenting cells and then phagocytosed and trafficked to the lysosome or phagolysosome of the cells.
• the mild acidic conditions found in lysosomes and phagolysosomes of APCs should cause the acetal group of the bisacryloyl acetal crosslinker to be hydrolysed thereby degrading the microgels. This acid hydrolysis of the bisacryloyl acetal crosslinker increases the pore size of the microgels allowing the entrapped bioactive material to diffuse out.
• This swelling of the microgels increases the osmotic pressure inside the cellular compartment which causes the cellular compartment to burst, thus releasing the bioactive material into the cytoplasm where it is exposed to the MHCI protein.
• the MHCI protein should then display the bioactive material on the cell surface of the antigen presenting cell and activate cytotoxic T lymphocytes (CTL) which can then recognize virus infected cells that display the bioactive material, thus targeting TCD + 8 immune response.
• CTL cytotoxic T lymphocytes
• the bisacryloyl acetal crosslinker of the invention is stable to basic conditions but hydrolyzes rapidly in acidic environments.
• a generic formula for the bisacryloyl acetal crosslinker (labeled as Compound I) is a general structure of R 2 CH(-OR ! ) 2 , where R 1 contains an acryl polymerizing group, and R 2 is a water solubilizing group. Acid degradable microgels can then be synthesized by copolymerizing this crosslinker with polymerizable groups in the presence of a bioactive material.
• the design of the bisacryloyl acetal crosslinker also reflects such factors as ease of synthesis, solubility, commercially available reagents, the type of hydrogel or microgel particle desired, the loading efficiency, dispersion of the particles, toxicity and the hydrolysis rates of the acetal linkage.
• Appropriate Acryloyl Groups (R 1 ) A key factor in choosing acryloyl groups is the chosen synthesis strategy of the crosslinker.
• Appropriate acryloyl groups (or acryl polymerizing group) that can be used for the bisacryloyl acetal crosslinker of this invention include but are not limited to, ethylacrylamides (a), methylacrylamides (b), acrylates (c), acrylamides (d), trifluoro-, tribromo-, trichloro- and triiodo- acetamides (e) and ethylamines (f).
• the acryloyl group (R 1 ) is an acrylamide, a substituted acrylamide, such as a methyl or ethyl acrylamide, acrylates or acrolein groups, substituted amides, and substituted vinyl groups.
• the present crosslinkers that are stable at higher than pH 7.0 but hydrolyze at a pH preferably about 5.
• the bisacryloyl acetal crosslinker should have an aqueous solubility of greater than 50mg/ml. This solubility is important to insure that the microgels will be polymerizable under inverse microemulsion conditions.
• Water solubilizing groups to use for R 2 to create the acetal linkage are compounds that contain polar groups and good leaving groups.
• Water solubilizing groups include but are not limited to, an, aryl, alkylaryl, alkoxy, aroxy or diaryl group, benzaldehyde or methoxybenzaldehyde.
• the acid degradable crosslinker of the invention has advantages over other types of linkages in the prior art.
• the crosslinker of the invention disclosed herein hydrolyzes on the timescale in which endosomes mature into lysosomes, in contrast to the extremely slow hydrolysis rates of cyclic ketals.
• the crosslinker in this invention uses an aromatic acetal as the acid degradable linkage.
• the aromatic acetal has two important advantages over ketals, (i) their hydrolysis rates can be controlled by adding substituent water solubilizing groups in their para position, and (ii) the aromatic portion of the acetal can also act as a functional group for conjugation to biodegradable polymerizable groups, such as dextrans.
• an autocatalytic bisacryloyl acetal crosslinker can be used to design novel types of microgels in which an amplification of the rate of release of bioactive material is provided by each cleavage step.
• a strategy for using an autocatalytic bisacryloyl acetal crosslinker involves the condensation of one molecule of a carbonyl compound, such as a benzaldehyde, with two molecules of a carboxylic acid or derivative thereof.
• Fig. 5A shows an bisacrylic benzaldehyde acetal crosslinker 530 obtained by incorporation of two acrylic acid moieties in an acetal like structure with benzaldehyde.
• hydrolysis of the autocatalytic crosslinker 530 under mild acidic conditions proceeds with release of two molecules of acrylic acid. Such release contributes to increase the acidity of the medium, thereby accelerating further hydrolysis. Amplification is desirable as it may contribute to faster release of the encapsulated bioactive material possibly enabling release from the normally less acidic endosomes or other compartments of cells.
• Preparation of microgels using such an autocatalytic crosslinker can also be carried out using inverse emulsion polymerization with suitable co-monomers including but not limited to, hydroxymethyl methacrylate or acrylamide.
• R 2 of the bisacryloyl acetal crosslinker is a benzaldehyde acetal with a para water solubilizing group to ensure that microgels made with the crosslinker hydrolyze rapidly after experiencing the pH 5.0 environment of acidic cellular compartments.
• the bisacryloyl acetal crosslinker becomes acid degradable because it has two ethers connected to the same carbon.
• the para water solubilizing group is electron donating and causes rapid hydrolysis.
• the water solubilizing groups can be positioned in the ortho- and meta- positions of the benzaldehyde, however, the ease of synthesis and availability of suitable starting reagents may make these crosslinkers more difficult to synthesize.
• water solubilizing groups include but are not limited to, an alkoxy, aroxy or diaryl group, benzaldehyde or methoxybenzaldehyde, having an ortho or para functional group such as triethylene glycol (triglyme), tetraethylene glycol (tetraglyme), polyethylene glycol, nitrochloroformate, dextrans, saccharides, sugars, and other carbohydrates, and combinations thereof.
• the size of the functional group should preferably have a molecular weight of less than 100,000.
• Linker groups including such groups as (-O-CH 2 -CH 2 -NH-C(O)-) or (-O-CH 2 -CH 2 -O-C(O)-O-) may be used to link these functional groups to the benzaldehyde acetal and aid in the addition and synthesis of these crosslinkers.
• the bisacryloyl acetal crosslinker has a benzaldehyde acetal with a para functional group selected from the group consisting of: hydrogen, aldehyde, dimethyl amine, methoxy, triethylene glycol, tetraethylene glycol, polyethylene glycol, and nitrochloroformate.
• the bisacryloyl acetal crosslinker can be hydrolyzed to release the contents entrapped in the microgels of the invention in a pH dependent manner.
• the bisacryloyl acetal crosslinker should preferably have a half-life at pH 5.0 of 5 minutes to 24 hours at 37°C, but a longer half life at pH 7.4 of at least 24 hours to 250 days.
• the crosslinker may be useful for the crosslinker to have a half-life at pH 5.0, 37°C of about 24 hours, and a half-life at pH 7.4, 37°C of about 250 days, in order to facilitate slow release of bioactive materials.
• the half-life of the crosslinker at pH 5.0, 37°C preferably be 5-30 minutes, and even more preferably be less than 5 minutes and a half-life at pH 7.4, 37°C of about 24 hours in order to quickly release the bioactive materials.
• the acceleration of the hydrolysis kinetics of a bisacryloyl acetal crosslinker from pH 7.4 to pH 5.0 is expected because the hydrolysis of the acetal is proportional to the hydronium ion concentration, which should increase between pH 7.4 and pH 5.0.
• the kinetics of acetal hydrolysis can be easily manipulated by introducing the appropriate electron withdrawing or donating groups and therefore it is possible to engineer acetal crosslinked hydrogels that have hydrolysis rates tailor-made for a given application.
• the hydrolysis kinetics of the bisacryloyl acetal crosslinker changes after its incorporation into the microgels.
• This change in the hydrolysis kinetics of the crosslinker is potentially due to two factors, (1) the steric effects of tethering the acetal moiety of the crosslinker into the particle, which in effect generates a cyclic acetal (cyclic acetals hydrolyze 10-100 times slower than straight chain acetals because of steric reasons) and (2) the diffusion of the hydronium ion into the microgels.
• a kinetic factor that may be taken into account when designing the bisacryloyl acetal crosslinker is the crosslinker' s speed of hydrolysis in solution.
• the bisacryloyl acetal crosslinker should preferably hydrolyze within 5-30 minutes at pH 5.0 at 37°C. This timescale is chosen because it is approximately the amount of time taken for a phagocytosed gel particle to be trafficked to cellular compartments such as lysosomes. In a preferred embodiment, these particles will degrade rapidly in the lysosome and cause lysosomal disruption.
• the crosslinker should hydrolyze fairly rapidly at a preferred range of pH 7.4 to 4.5 and even more preferably between pH 6.8 to 4.5. 6. Synthesis of the Crosslinker i a general embodiment, the bisacryloyl acetal crosslinker has the general structure of R 2 CH(-OR 1 ) 2 , which can be made by first synthesizing the R 2 water solubilizing group.
• the strategy for synthesis is reacting the water solubilizing group with an amino alcohol that has its amine group protected with an acid stable protecting group, such as a trifluoroacetamide, to form an acetal.
• the protected amines can then be deprotected and reacted with acryloyl chloride to generate the bisacrylamide acetal crosslinker.
• an alcohol is used having no amino group, it can be reacted with an acryloyl chloride to make an acrylate.
• use of an acrylate may raise some solubility issues as acrylates tend to be 100-fold less soluble in the aqueous phase as acrylamides.
• the strategy for synthesis involves the condensation of one molecule of the water solubilizing group with two molecules of a carboxylic acid or derivative thereof to generate an autocatalytic bisacryloyl acetal crosslinker.
• the crosslinker is selected from the group consisting of: bisacrylamide methoxybenzaldehyde acetal crosslinker 110 (as shown in Fig. 4), bisacrylic benzaldehyde acetal crosslinker 530 (as shown in Fig. 5A), bisacrylamide triglyme acetal crosslinker 606 (as shown in Fig. 6), bisacrylamide tetraglyme acetal crosslinker 710 (as shown in Fig. 7), bisacrylamide nitrochloroformate acetal crosslinker 836 (as shown in Fig. 8A) and bisacrylamide dextran acetal crosslinker 920 (as shown in Fig. 9C). Acid degradable microgels can then be synthesized by copolymerizing this bisacryloyl acetal crosslinker with polymerizable groups in the presence of a bioactive material.
• Microgel Particles for the Delivery of Bioactive Materials Microgels made with the bisacryloyl acetal crosslinker should efficiently entrap bioactive material with a comparable loading efficiency.
• the microgel particle size may vary between 0.1-10 ⁇ m and exhibit a loading efficiency of at least 40% bioactive material encapsulation, more preferably at least 50% loading efficiency and even more preferably at least 54% loading efficiency.
• Appropriate Polymerizable Groups for Gel Particles include acrylic polymers such as, acrylamides, methacrylamides, methacrylates, and acrylates.
• the polymerizable groups are acrylamides or methacrylamides.
• Appropriate biocompatible polymerizable groups that can be used for the microgels of this invention include biocompatible polymers including but not limited to, dextrans, saccharides, mannoses, sugars, carbohydrates, nucleic acids, oligonucleotides, amino acids, polypeptides, lipids and combinations thereof.
• the biocompatible polymerizable group is dextrans up to 100,000 MW, more preferably up to 10,000 MW.
• the biocompatible polymerizable group is conjugated to the functional group X of R of the Compound I bisacryloyl acetal crosslinker before synthesis of the microgels.
• the invention contemplates entrapping such bioactive materials including but not limited to, nucleotides, polynucleotides, ribonucleotides, amino acids, peptides, proteins, antigens, plasmid DNA, growth factors and hormones, interleukins, immunostimulatory agents, drugs, vaccines, neuromodulatory agents such as neurotransmitters, stimulatory and adrenergic agents, enzymes, proteases, anticancer and antitumor agents, imaging agents, diagnostic agents, antiviral agents and antibacterial agents.
• bioactive materials including but not limited to, nucleotides, polynucleotides, ribonucleotides, amino acids, peptides, proteins, antigens, plasmid DNA, growth factors and hormones, interleukins, immunostimulatory agents, drugs, vaccines, neuromodulatory agents such as neurotransmitters, stimulatory and adrenergic agents, enzymes, proteases, anticancer and antitumor agents, imaging
• the bioactive material is selected from the group consisting of: nucleotides, polynucleotides, proteins, immunostimulatory agents, vaccines, antigens, anti-viral agents, protein antigens, anticancer agents and antitumor agents.
• bioactive materials can be conjugated with a carrier molecule or they can be conjugated to a polymerizable group and copolymerized into the microgels.
• the linkage between the polymerizable group and the bioactive molecule can be designed to be cleaved under various physiological conditions.
• the bioactive material can also be adsorbed onto the surface of the microgels, or reacted to the surface of the microgels. 3.
• microgels can be synthesized by inverse microemulsion polymerization according to the procedure described by Kriwet, B.; Walter, E.; Kissel, T.; J. Control Release, 1998, (56), 149-158, which describes synthesis of bioadhesive poly(acrylic acid) nano- and microparticles using an inverse emulsion polymerization method for the entrapment of hydrophilic drug candidates.
• a key issue in the synthesis of microgels by inverse emulsion polymerization is the aqueous solubility of the acryloyl groups and polymerizable groups. The solubility of both the acryloyl groups and polymerizable groups are very important as all of the polymerizable components in an inverse emulsion polymerization must be sufficiently water soluble.
• a small amount of water is dispersed into an organic phase and stabilized by surfactants. Sonication before polymerization for about 5 minutes will insure the correct particle size, which will cover a range of sizes, within the range of about 100 nm -10 ⁇ m, preferably 100 nm - 5 ⁇ m.
• the polymerizable groups and the bisacryloyl acetal crosslinker are then polymerized in the aqueous phase in the presence of the bioactive material and an initiator molecule or radical source. Since polymerization is initiated and contained within water droplets, mainly spherical crosslinked microgel particles containing entrapped bioactive material are produced. To adjust particle size, either longer sonication time or larger surfactant concentration will decrease the microgel particle size.
• the bisacryloyl acetal crosslinker exhibits improved performance in forming microgel particles in hexane/water versus chloroform-toluene/water is potentially explained by the lower solubility of the crosslinker in hexane versus chloroform-toluene.
• the bisacryloyl acetal crosslinker has water/hexane partition ratio of 10,000:1.
• the water/toluene-chloroform partition ratio is only 1:1, suggesting that in the water/chloroform- toluene polymerizations, a large fraction of the crosslinker is lost in the organic phase.
• the organic phase is most preferably hexane and the surfactants used are preferably TWEEN TM 80, SPAN T 80, dioctyl sulfo succinate (AOT) and Brij or combinations thereof. More preferably the surfactant used is a 1:3 ratio of TWEEN TM 80 / SPAN TM 80. Furthermore, it is important that the surfactants used for synthesis be neutral and FDA approved for human use. Neutral, biocompatible surfactants are preferred for the synthesis of bioactive material-loaded microgel particles because of their reduced interactions with proteins and lower toxicity. 4. Loading and Loading Efficiency of Entrapped Bioactive Materials
• the bisacryloyl acetal crosslinker can affect that loading efficiency and the amount of bioactive material entrapped in the microgels of the invention.
• Loading efficiency is the amount of bioactive material that is entrapped within the gel particles as compared to the total starting amount of bioactive material placed in the polymerization reaction.
• the loading efficiency of the microgel particles of the invention should not appreciably change with the crosslinking ratio, however, the water solubilizing groups can change the loading and encapsulation efficiencies.
• the loading efficiency is different from the amount of protein encapsulated in a single particle. It is estimated that approximately 1 million protein molecules of about 50 kD size can be encapsulated in a microgel. This number comes from assuming the microgel has a density of 1, a radius of 0.5 microns, and are composed of 10% protein by weight.
• the microgel particles of the invention should have at least a 20% loading efficiency, more preferably 40% loading efficiency, even more preferably at least 50% loading efficiency, and most preferably more than 55% loading efficiency.
• the loadings and efficiencies of the microgel particles should be comparable to other microparticle systems which have efficiencies purported to be about 1-2 ⁇ g DNA/mg polymer for 500 nm PLGA particles.
• efficiencies purported to be about 1-2 ⁇ g DNA/mg polymer for 500 nm PLGA particles.
• the loading efficiencies for the amount of DNA material entrapped in microgel particles of the preferred embodiment should preferably be at least 40%, more preferably at least 50% and even more preferably at least 54%.
• the loading efficiencies for the amount of protein entrapped in microgel particles of the preferred embodiment should be at least 20%, preferably at least 40%, more preferably around 50%.
• the loading efficiency and the amount of bioactive material entrapped is an important aspect in light of such factors as the amount of bioactive material needed to be delivered to the target for an effective dose and the amount of available bioactive material.
• a major drawback in previous therapeutics and vaccines is there is often difficulty in obtaining large enough amounts of the therapeutic composition of bioactive material for production. Therefore, it is a goal of the invention to make microgels with high loading efficiencies so as to lower the starting amount of bioactive material required at the beginning of polymerization.
• the release of bioactive materials from the loaded gel particles made with the bisacryloyl acetal crosslinker can be first measured at various mild pHs at 37° C, in aqueous solutions.
• the crosslinker should hydrolyze within 5 minutes to 24 hours at pH 5.0 and have a much slower hydrolysis rate at pH 7.4.
• these microgels will degrade rapidly in the lysosomes and cause lysosomal disruption. Therefore, in a preferred embodiment, at a more acidic pH 5.0, encapsulated bioactive materials should preferably be 80% released from the microgel particles within 6 hours, preferably completely released from the microgel particles within 12 hours, more preferably within 8 hours, and even more preferably within 6 hours. At pH 7.4, the release of entrapped bioactive materials 40 should be significantly slower, taking up to 150 hours for the microgel particles to completely release their contents. The molar ratio between the concentration of polymerizable groups and the bisacryloyl acetal crosslinker affects the rate of bioactive material released from the gel particles.
• the microgels of the present invention have 1- 20 mole percent crosslinking, between 1% - 12.8% crosslinking, and most preferred between 1% - 3% crosslinking, but sufficient to physically trap the bioactive molecule within the microgel.
• a molar ratio of 9: 1 acrylamide bisacryloyl triglyme-acetal crosslinker (1.6% crosslinking) results in a linear release of bioactive material, with nearly 80% released at 300 minutes.
• a ratio of 4:1 of acrylamide to bisacryloyl triglyme-acetal crosslinker (3.5% crosslinking) results in a steeper initial increase in release of bioactive material with a slower increase from 100-200 minutes and an second steep increase from 200-300 minutes.
• a small molar ratio of 1:1 acrylamide/ bisacryloyl triglyme-acetal crosslinker (12.8% crosslinking) results in a very slow release of 20% of the bioactive material in the first 175 minutes and a steep release of bioactive material.
• the amount of crosslinker can be increased or decreased depending on the desired rate of release of bioactive material.
• 1-3% crosslinking may be preferred, not only for its steady linear release of bioactive material, but also because of the crosslinker' s effects upon other factors such as toxicity, loading efficiency, and amount of T- cell activation.
• increased amounts of crosslinker may be more preferred, such as in a case for example, where the crosslinker size is small.
• microgels release their bioactive material payload into the cytoplasm of cells upon lysosomal disruption. Higher loading capacity of the gel particles may also lead to greater antigen presentation of the encapsulated bioactive material. Li the antigen presentation assay described by Sanderson, S.; Shastri, N. in Inter.
• Antigen presenting cells display the peptide having the sequence, SHNFEKL, upon phagocytosis of ovalbumin. These cells were engineered to transcribe ⁇ -galactosidase when in the presence of antigen presenting cells displaying the SHNFEKL peptide. ⁇ -galactosidase then liberates chlorophenol red from the chlorophenol red ⁇ galactoside that is present in solution.
• the bioactive loading capacity and efficiency should lead to an absorbance of preferably at least 0.15, more preferably 0.2, and most preferably more than 0.25, using the antigen presentation assay described by Sanderson, S.; Shastri, N. in Inter. Immun. 1994, 6, 369-376.
• a preferred basic minimal level of antigen presentation that the particles should effectuate is about 50% T-cell activation as the minimum level of T-cell activation.
• Efficient microgels should need approximately 500 micropaticles per antigen presenting cell.
• the level beyond which the starting amount of bioactive material and micrgels compared to the amount of antigen presentation is inefficient and unpreferred is considered about 5mg/ml of microgels to generate a 100% T cell activation. This level is inefficient and unpreferred because of the potential toxicity involved with the delivery vehicles.
• the target antigen presenting cells should preferably exhibit at least 50% viability after 24 hours of incubation with the gel particles of the mvention, more preferably at least 70% viability after 24 hours, even more preferably at least 80% viability and most preferably more than 90% viability after 24 hours according to the MTT assay as described above and in Example 14.
• microgel particles that easily and safely excreted by the body after being degraded in the acidic cellular compartment.
• the microgel particles degrade into linear polymer chains are 100,000 daltons or less.
• dextran microgels are one preferred embodiment because dextrans degrade into chains of 10,000 daltons or less.
• the microgels of the present invention would have applications in vaccine therapeutics and disease prevention.
• Protein loaded microgels could be injected as an intramuscular injection to a patient, stimulating phagocytosis by macrophages and antigen presenting cells. After being sequestered in lysosomes, the acid degradable linkage of the bisacryloyl acetal crosslinker would hydrolyze, releasing the protein antigen, and cause the lysosome to swell and then burst, thereby releasing the lysosome contents into the cellular cytoplasm. Once the protein antigen is released into the cytoplasm, MHCI proteins can then bind the protein antigen and present the protein antigen on the cell membrane. These cells would then initiate the cytotoxic T lymphocyte immune response against pathogens from which the protein antigen came from.
• the gel particles of the invention would be particularly useful in combating infections that need a strong cytotoxic T lymphocyte response, including diseases such as HIV/ AIDS and Hepatitis C infections.
• antigens which can be used as bioactive material and entrapped in the microgels of the present invention, include but are definitely not limited to, the TAT protein from HTV, the ENV protein from HIV, the Hepatitis C Core Protein from the Hepatitis C virus, the prostatic acid phosphatase for prostate cancer and the protein MART-1 for melanoma.
• the microgels of the invention would be used for gene therapeutics.
• the microgels of the invention would be especially suited for this application. Once a polynucleotide is delivered by the microgels to the cytoplasm, the polynucleotide can undergo translation into a protein. This has the potential, then, to make proteins that are not normally produced by a cell.
• the bioactive material would be a plasmid that encodes for a protein or antigenic peptide initially. For example, one would use a plasmid that encodes for a protein that would display antigens for cancer.
• Plasmid DNA encapsulated in the microgels of the invention and delivered to the cytoplasm of phagocytic cells, has been shown to be active and still able to transfect cells.
• plasmid DNA has the added characteristic of generating an immune response because plasmid DNA is generated from bacteria.
• Bacterial DNA has two major differences compared with vertebrate DNA: 1) bacterial DNA has a higher frequency of CG dinucleotides in the sequence (1/16 dinucleotides in microbial DNA are CG pairs, but only 25% of that is observed in vertebrate DNA); and 2) bacterial DNA is unmethylated as compared to vertabrate DNA which is often methylated. Vertebrate systems will recognize the plasmid DNA then as being foreign, and the cell will react as for a bacterial infection.
• the plasmid DNA used as the bioactive material would have an added interleukin sequence.
• Interleukins are secreted peptides or proteins that mediate local interactions between white blood cells during immune response (B. Alberts et al, Molecular Biology of the Cell, 4th ed., Garland Science, 2002). Different interleukins (e.g. IL-12, IL-2) will direct the type of immune response that is generated.
• T H 1 response is characterized by a CTL response; a T H 2 response is characterized by antibody production.
• interleukin-2 or 12 IL-2 or IL-12
• IL-2 or IL-12 interleukin-12
• immunostimulatory groups include but are not limited mannose, plasmid DNA, oligonucleotides, ligands for the Toll receptors, interleukins and chemokines.
• T-cells activate B-cells to secrete H ⁇ terleukin-6 (IL-6) to stimulate B cells into antibody-secreting cells. It has been shown by protestopoulos, V.; McKenzie, I. F. C.
• oligonucleotides (approximately 12-75 bases in length) can be used for immunostimulation as well as for their antisense activity.
• oligonucleotides are too small to remain encapsulated inside the microgels of the invention, they must be conjugated to a polymerizable group and then later released.
• One way to solve this problem would be to attach the oligonucleotides to a polymerizable group through an acid degradable linkage similar to creating a second crosslinker, whereby acid hydrolysis in the acidic conditions will release the oligonucleotide.
• the oligonucleotides can be conjugated to a large macromolecule, such as dextran through an acid degradable linkage which is then physically entrapped in the microgels.
• microgels of the invention can be suspended or stored in a conventional nontoxic vehicle, which may be solid or liquid, water, saline, or other means which is suitable for maintaining pH, encapsulation of the bioactive material for an extended period of time, sufficient dispersion or dilution of the microgels and the overall viability of the microgels for their intended use.
• a conventional nontoxic vehicle which may be solid or liquid, water, saline, or other means which is suitable for maintaining pH, encapsulation of the bioactive material for an extended period of time, sufficient dispersion or dilution of the microgels and the overall viability of the microgels for their intended use.
• the microgel particles of the invention are stored in dry state (vacumm dried) and stored at 4°C for several months.
• the microgels can be dispersed in buffer and sonicated or vortexed for a few minutes to resuspend into solution when needed.
• the microgels should be vortexed or sonicated for a sufficient amount of time to resuspend the microgels evenly in solution, however, not too long as vortexing and sonication can also damage some proteins and bioactive material.
• the microgel particles are ready for delivery upon visual determination that the microgels are sufficiently dispersed in solution.
• the solution should be opaque with no visible aggregates floating.
• the loaded microgels of the invention can be administered by various suitable means to a patient, including but not limited to parenterally, by intramuscular, intravenous, intraperitoneal, or subcutaneous injection, or by inhalation.
• the delivery of the microgels to a patient is preferably administered by injection once but does not preclude the necessity for multiple injections that would be required to illicit the desired level of immune response.
• the amount of microgels needed to deliver a pharmaceutically effective dosage of the bioactive material to effect the CTL response in a patient will vary based on such factors including but not limited to, the crosslinker and polymerizing group chosen, the protein loading capacity and efficiency of the gel particles, the toxicity levels of the biodegraded particles, the amount and type of bioactive material needed to effect the desired response, the subject's species, age, weight, and condition, the disease and its severity, the mode of administration, and the like.
• One skilled in the art would be able to determine the pharmaceutically effective dosage.
• the amount of bioactive material that could be administered by the microgels of the invention is from 1 ng to more than lgram quantities.
• FIG. 3 an exemplary process for the synthesis of a microgel particle for pH-dependent degradation is shown.
• the polymerization reaction is carried out by inverse microemulsion, initiated by an initiator such as TMEDA and diammonium sulfate and carried out in the presence of bioactive particle 40.
• the bisacrylamide methoxybenzaldehyde acetal crosslinker 110 remains intact and the release of entrapped bioactive material 40 is significantly slower or not at all.
• the acetal group hydrolyses and increases the pore size of microgels made with it, releasing the entrapped bioactive materials 40, and degraded particles, in the form of multiple polymer chains 140 and a small molecule aldehyde 130.
• the bisacrylamide methoxy benzaldehyde acetal crosslinker 110 was synthesized in two steps. The first step was acetal formation with hydroxy- trifluoroacetamide and methoxy benzaldehyde using the procedure of Roelofsen et al., Recueil, 90, 1141-1152 (1971, which gave a 70% yield of bistrifluoroacetamide methoxy benzaldehyde acetal 410 after chromatography.
• a strategy for synthesis of an autocatalytic bisacryloyl acetal crosslinker involves the condensation of one molecule of carbonyl compound such as a benzaldehyde with two molecules of a carboxylic acid or derivative thereof.
• R l (c) of Fig. 2
• R 2 aryl-H.
• Figure 5 A shows a bisacrylic benzaldehyde acetal crosslinker 530 obtained by incorporation of two acrylic acid moieties in an acetal like structure with benzaldehyde.
• para methoxy benzaldehyde 510 is reacted with acrylic anhydride 520 in the presence of sulfuric acid to form the autocatalytic bisacrylic benzaldehyde acetal crosslinker 530.
• an alternative synthesis is to react dibromo-toluene with sodium acrylate 550 to form the crosslinker 530.
• Preparation of microgel particles using this release-amplified crosslinker can also be carried out using inverse emulsion polymerization with suitable co-monomers such as hydroxymethyl methacrylate or acrylamide.
• suitable co-monomers such as hydroxymethyl methacrylate or acrylamide.
• a more hydrophilic acid degradable crosslinker, containing a hydrophilic triethylene glycol (triglyme) moiety is shown. Since the crosslinker of Example 2 is relatively hydrophobic, it was hypothesized that decreasing its hydrophobicity would improve its performance since inverse emulsion polymerizations are very sensitive to the hydrophobic/hydrophilic balance of the reactants. This modification dramatically increased the hydrophilicity of the crosslinker making it compatible with a variety of inverse emulsion polymerization procedures.
• the bisacrylamide triethylene glycol acetal crosslinker (606) or triglyme crosslinker as shown in Figure 6 was synthesized in four steps on a multigram scale.
• the first step involved the preparation of l-chloro-3,6,9-trioxadecane (603) using the procedure of Loth, H. & Ulrich, F. (1998) J. Control Release. 54, 273.
• Compound 603 was then used to alkylate hydroxy benzaldehyde (605) and produce p-( 1,4,7, 10- Tetraoxaundecyl)benzaldehyde (604).
• Compound 603 was chosen as the alkylating agent since it can be easily synthesized on a large scale (100 grams) enabling the preparation of 4 on a 20 gram scale (70% yield), using potassium carbonate as the base and 18-crown-6 as the phase transfer catalyst. Hydroxy-benzaldehyde could also be alkylated using l-tosyl-3,6,9- trioxadecane and l-bromo-3,6,9-trioxadecane but these approaches were discontinued because significantly lower yields of the desired product were obtained.
• Compound 604 was converted to an acetal (605) by reaction with N-(2- hydroxyethyl)-2,2,2-trifluoroacetamide.
• a potential problem with acetal formations is the separation of the product from residual alcohol. The alcohol is generally used in 4-6 fold molar excess over the aldehyde, and even high yielding reactions leave 2-4 molar equivalents of the alcohol to be removed.
• Initial attempts at purifying the acetal product 605 from unreacted N-(2-hydroxyethyl)-2,2,2-trifluoroacetamide using flash chromatography were unsuccessful. However, 605 could be purified by crystallization from ethyl acetate/hexane, allowing for its synthesis on a multigram scale.
• the final bisacrylamide triethylene glycol acetal crosslinker was obtained by cleaving the trifluoroacetyl groups on 605 in 6 M NaOH/Dioxane followed by reaction of the resulting diamine with an excess of acryloyl chloride.
• Final purification of the corsslinker was achieved by crystallization from ethyl acetate/hexane. l-Chlor ⁇ "3,6,9-trioxadecane (603).
• Spectroscopic data agreed with those reported in the literature. 13 C NMR(CDC1 3 ): ⁇ 42.16, 58.30, 69.92, 69.96, 69.98, 70.70, 71.30. Anal. Calcd.
• N,N'-Bis1rifluoroacetyl-di-(2-aminoethoxy)-[4-(l,4,7,10- tetraoxaundecyl)phenyl]methane (605).
• Aldehyde 604 (3.60 g, 13.4 mmol, 1 equiv) and N- (2-hydroxyethyl)-2,2,2-trifluoroacetamide (15.0 g, 95.5 mmol, 7.1 equiv) were dissolved in dry THF (50 mL).
• p-Toluenesulfonic acid (0.360 g, 2.09 mmol, 0.16 equiv) and 5 A molecular sieves (50 g) were added.
• N,N'-Bisacryloyl-di-(2-aminoethoxy)-[4-(l,4,7,10- tetraoxaundecyl)phenyl]methane (606).
• Compound 605 (4.0 g, 7.1 mmol, 1 equiv) and 6 M NaOH (30 mL) were added to dioxane (20 mL) and the reaction mixture was stirred at room temperature for 7 h. Complete removal of the acetamide groups was determined by TLC using ninhydrin staining. Upon completion, the reaction mixture was cooled to 0 °C and triethylamine (3 mL) was added.
• This crosslinker has properties, as compared to the other crosslinkers disclosed herein, including, but not limited to, increased protein loading and loading efficiency, better dispersability in solution, and higher T-cell activation achieved.
• a key aspect of the bisacryloyl acetal crosslinker is its hydrolysis kinetics.
• the crosslinker is designed to be stable at the physiological pH of 7.4 but it undergoes rapid hydrolysis at acidic pHs. This is demonstrated by measurements performed with the bisacrylamide methoxybenzaldehyde acetal crosslinker 110 at pH 5.0 and at pH 7.4. At pH 5.0, the crosslinker hydrolyzes rapidly, with a half-life of 5.5 minutes, whereas at pH 7.4 the half-life is 24 hours.
• a stock solution of the bisacrylamide methoxybenzaldehyde acetal crosslinker (10 mg/mL) in THF was prepared and 10.5 ⁇ L (lxlO 4 mol/L) was added to a 3.0ml PBS solution at either pH 5.0 or 7.4, in a spectrophotometer cuvette.
• the hydrolysis of the acetal was monitored by measuring the absorbance of the 4-methoxybenzaldehyde, produced by the acetal hydrolysis, at 280 nm.
• pH 5.0 95% hydrolysis was complete in 20 minutes, with about 50% hydrolysis in less than 10 minutes.
• Microgel particles were synthesized by inverse microemulsion polymerization, according to the procedure described by Kriwet, B.; Walter, E.; Kissel, T.; J. Control Release, 1998, (56), 149-158.
• a key issue in the synthesis of microgels by inverse emulsion polymerization is the aqueous solubility of the monomers.
• Several different emulsion polymerization procedures were attempted with the bisacrylamide acetal crosslinker, using different organic phases and surfactant blends. Inverse polymerizations with toluene/chloroform as the continous phase and pluronic F-68 as the surfactant were unsuccessful.
• the improved performance of the bisacrylamide methoxy benzaldehyde acetal crosslinker 110 in hexane water versus chloroform-toluene/water is potentially explained by the lower solubility of the crosslinker in hexane versus chloroform-toluene.
• the bisacrylamide methoxy benzaldehyde acetal crosslinker 110 has water/hexane partition ratio of 10,000: 1 , in contrast the water/toluene-chloroform partition ratio is only 1:1, suggesting that in the water/chloroform-toluene polymerization a large fraction of the crosslinker is lost in the organic phase.
• Example 11 The following protocol illustrates the preparation of the present microgel particles encapsulating a bioactive material (albumin). These particles are discussed further in connection with Example 11.
• Table 1 of Example 11 sets forth the components of three different microgel particles.
• microgel particles with crosslinking ratios ranging from 1.6%-12.8% were prepared using this inverse emulsion polymerization procedure with bisacrylamide triglyme acetal crosslinker 606 of Example 4 and acrylamide.
• the organic phase of the polymerization consisted of 5 mL of hexane containing 150 mg of a 3: 1 weight ratio of SPAN TM 80 and TWEENTM 80.
• the aqueous phase of the polymerization consisted of 125, 200, or 225 mg of acrylamide and 25, 50 or 125 mg of the bisacrylamide triglyme acetal crosslinker of Example 4 (with a combined weight of 250 mg), dissolved in 0.5 ml of sodium phosphate buffer pH 8.0300 mM sodium phosphate, 12 mg of the free radical initiator potassium peroxodisulfate and 5.4 or 5.7 mg ovalbumin.
• the aqueous and organic phases were deoxygenated with nitrogen.
• the mixture was centrifuged at 2800 rpm for 10 minutes and the solvent was decanted off.
• the microgels were carefully washed with hexane (2 x 20 mL), acetone (4 x 20 mL) and isolated by centrifugation at 2,800rpm for 10 minutes.
• the recovered microgels were vacuum dried overnight and analyzed by scanning electron microscopy (WDX ISI-dsl30C, Microspec Corp.) at 15 kV.
• a scanning electron microscopy (SEM) image of the particles (not shown) showed that the particle size varied between 200 nm and 500 nm in the dry state. This size distribution is suitable for protein delivery to APCs, which internalize particles between 200 nm-5 ⁇ m by phagocytosis.
• a bisacrylamide nitrochloroformate acetal crosslinker 836 can be synthesized by the synthesis steps as shown in Fig. 8 A.
• the synthesis of the bisacrylamide nitrochloroformate acetal crosslinker 836 was accomplished in four steps.
• the first step was alkylation of hydroxy benzaldehyde 810 with bromo-ethylacetate 820, using potassium carbonate and 18-6 crown as the base.
• the product was purified by a small silica gel column, and this synthesis could be performed on a 20 gram scale.
• the second step was acetal formation between hydroxy-ethyl trifluoroacetamide 830 and the benzaldehyde acetate 830 from previous, using p-toluene sulfonic acid as a catalyst.
• the bistrifluoroacetamide methoxyphenyl-ethyl acetate 834 was then deprotected in 6N NaOH and reacted with acryloyl chloride, the reaction product was purified by crystallization from ethyl acetate, to give the hydroxyl compound 835. This reaction needs to be performed in 6N NaOH otherwise the hydroxyl will react with the acryloyl chloride.
• the compound 835 was converted to the bisacrylamide nitrochloroformate acetal crosslinker 836 by reacting with p ⁇ r -nitrochloroformate, in the presence of triethyl amine.
• the crosslinker 836 was purified by crystallization from ethyl acetate.
• Dextran microgel particles were made using the bisacrylamide nitrochloroformate acetal crosslinker 836 of Example 8 using inverse microemulsion polymerization as described in Example 7.
• the bisacrylamide nitrochloroformate acetal crosslinker 836 was modified to the dextran acetal crosslinker 840 by introducing an amine handle on dextran and then reacting this activated dextran 838 with the crosslinker 836.
• the amine was introduced onto the dextran by activating the dextran hydroxyls with p ⁇ r -nitrochlorofomate and then reacting it with diamino-diethylene glycol.
• H-NMR indicated that 1 out every six hydroxyls were functionalized with the amine handle.
• the purification of the dextran products was performed by precipitating the reaction in ethanol.
• the final dextran acetal crosslinker 840 was synthesized by reacting the amine functionalized dextran 838 with the crosslinker 836, the product 840 was purified by precipitation in ethanol and size exclusion chromatography.
• H-NMR and UN. spectroscopy indicated that approximately 1 out 6 of the sugars reacted with the crosslinker 836.
• dextran microgels were made under inverse microemulsion polymerization conditions using SPAN 80/TWEEN 80 as surfactants and hexane as the oil phase.
• S 2 O 8 K 2 potassium persulfate
• 300 mg of the bisacrylamide-dextran-acetal crosslinker 840 in the presence of 1 mg DNA, copolymerized to entrap the DNA and formed loaded dextran microgels which are biodegradable as shown in Fig. 10.
• An SEM image of the microgels showed that the shape of the dextran microgels are not spherical, but nevertheless are individual microparticles.
• the number of dextran molecules in this example should preferably be between 3 to 555 sugar molecules, with a MW of no more than 100,000.
• EXAMPLE 10 Synthesis of Bisacrylamide-Dextran-Acetal Crosslinker
• a bisacrylamide-dextran-acetal crosslinker 920 can be synthesized by the synthesis steps as shown in Fig. 9A-9B.
• hydroxy benzaldehyde 902 is reacted with 1,3 bromo-propyl-chloride in the presence of K 2 CO 3 , 18-6 Crown Ether, and THF, resulting in benzaldehyde-4-methoxy-propyl chloride 844 at 30% yield.
• the azide is formed by reacting compound 904 with NaN 3 , DMF at 90-100°C, yielding azidopropyl benzaldehyde 906 in 70% yield.
• the acetal linkage is made by reacting the second intermediate, azidopropyl benzaldehyde 906, with (2) molecules of hydroxy-ethyl trifluoroacetamide, in the presence of p-toluenesulfonic acid, THF, using 5 A molecular sieves to yield the bistrifluoroacetamide methoxyphenyl-propyl-azide-acetal 908 at 49% yield.
• This intermediate azide-acetal 908 is then reduced to an amine acetal by PPh 3 , THF, TEA, H 2 O to yield a bistrifluoroacetamide amine acetal 910.
• the bistrifluoroacetamide amine acetal 910 is then reacted with p ⁇ r -nitrochloroformate-activated dextran 914 in DMSO to yield a dextran- trifluoroacetamide acetal.
• the activated dextran 914 is prepared as described in Example 9, wherein an amine handle on dextran was introduced to produce activated dextran 838.
• the amine was introduced onto the dextran by activating the dextran hydroxyls with para- nitrochlorofomate and then reacting it with diamino-diethylene glycol. H-NMR indicated that 1 out every six hydroxyls were functionalized with the amine handle.
• the purification of the dextran products was performed by precipitating the reaction in ethanol. The product is then purified by ether/ethanol precipitation and gel permeation chromatography to yield a bistrifluoriacetamide-dextran-acetal 916.
• the bisacrylamide dextran acetal 920 was synthesized in two steps by first reacting the bistrifluoriacetamide-dextran-acetal 916 with K 2 CO 3 , MeOH and H 2 O to yield a bisamine-dextran-acetal 918. The addition of pyridine and pH 10 buffer in the presence of acryloyl chloride to maintain pH then yields a bisacrylamide-dextran-acetal crosslinker 920.
• dextran microgels can be made using the inverse microemulsion polymerization conditions of Example 7.
• a radical source like potassium persulfate
• the crosslinker will polymerize and trap the bioactive material 40 and form loaded dextran microgels which are biodegradable as shown in Fig. 10.
• the number of dextran molecules attached should preferably be between 3 to 555 sugar molecules, with a MW of no more than 100,000.
• the bisacryloyl acetal crosslinker used to synthesize acid degradable protein loaded microgels influences the loading efficiency of the microgels.
• the bioactive material is physically entrapped in the microparticle by polymerizing the polymerizable groups and crosslinker in the presence of the bioactive material.
• a key parameter in the synthesis of protein-loaded microgels is their "pore size", which needs to be smaller than the radius of the protein or other bioactive material for efficient encapsulation.
• Ovalbumin was chosen as the model protein for the encapsulation studies because numerous immunological assays have been developed for this protein.
• Ovalbumin labeled with Cascade Blue was encapsulated in microgels containing 1.6, 3.5 and 12.8 mole percent of bisacrylamide triglyme-acetal crosslinker 606.
• the results of the protein encapsulation experiment are listed in Table 1, with protein loadings varying from 9-1 l ⁇ g of protein per mg of microgel. Based on the starting protein monomers ratio, this represents about 50% encapsulation efficiency. Protein Encapsulation Measurements. 2 mg of each microgel particle sample
• the protein concentration of each microgel sample was calculated by fitting the emission to a calibration curve made from known concentrations of Cascade Blue labeled Ovalbumin.
• the loading efficiency measurements may be lower than actual amount because there may protein entrapped in microgels that do not centrifuge down and thus cannot be recovered.
• the encapsulation efficiency obtained with the bisacrylamide triglyme-acetal crosslinker was similar to that observed by others for the encapsulation of ovalbumin in non- degradable microgels composed of 2.5 mole percent methylene bisacrylamide and 97.5 mole percent acrylamide (O'Hagan, D. T.; Palin, K.; Davis, S. S.; Artursson, P.; Sjoholm.; Vaccine. 1989, (7), 421-424.).
• the encapsulation efficiency of the microgel particles made with the bisacrylamide triglyme-acetal crosslinker did not change appreciably with the crosslinking ratio as shown by the amounts of encapsulated albumin per mg of microgel particle ( ⁇ g/mg) in Table 1.
• the protein loading efficiency of the gel particles polymerized with 1.6% crosslinking with the bisacrylamide triglyme-acetal crosslinker 606 of Example 4 was compared to the protein loading efficiency of the microgel particles polymerized with the 1.6% of the bisacrylamide tetraglyme-acetal crosslinker 710 of Example 5.
• the crosslinkers were copolymerized with acrylamide in a PBS buffer containing the fluorescently labeled protein FTTC-Albumin (lmg/ml). Table 2 lists the resulting amount of ovalbumin encapsulated per milligram of microgel particles.
• EXAMPLE 12 pH Dependant Release of Encapsulated Bioactive Material by Hydrolysis
• the bisacrylamide triglyme acetal crosslinker 606 was used to synthesize acid degradable protein loaded microgels according to the conditions described in Example 7. 2 mg of each microgel sample from Table 1 containing Cascade Blue labeled Ovalbumin was dispersed in 0.5 mL of pH 8.0300mM sodium phosphate buffered water by sonication for 5 minutes. The microgel samples were centrifuged for 5 minutes and the supernatant was ' pipetted off to remove any unbound protein.
• the recovered pellet was then redispersed into either 300mM acetic acid buffered water (pH 5.0, 500 ⁇ L) or 300mM sodium phosphate buffer (pH 7.4, 500 ⁇ L).
• the solutions were incubated at 37 °C in a heating block for each time point.
• the percentage of protein released at a given time point was determined by centrifuging the microgel sample for 5 minutes, isolating the supernatant from the pellet and comparing the fluorescence of the supernatant (released protein) with that of the pellet (protein still in microgels), excitation at 405nm, emission at 460nm.
• the recovered pellet was hydrolyzed in pH 1.6300mM acetic acid, before measuring its fluorescence.
• a proper control would be to compare the amount presented by the gel particles of the invention when incubated with the SIINFEKL peptide which is directly displayed on the antigen presenting cells and not delivered to the cytoplasm of the cells first.
• a maximum absorbance of 0.25 is observed with the SHNFEKL peptide, which results in 100% T-cell activation.
• the particles made with a 1:1 ratio of acrylamide to the bisacrylamide tetraglyme crosslinker of Example 5 shows an absorbance close to that of 0.25 at which 100% T-cell activation occurs.
• Ovalbumin encapsulated in the microgels is several orders of magnitude more efficient than free ovalbumin at inducing the activation of CTLs, for example, 1 ⁇ g/ml of ovalbumin encapsulated in the microgels gives T cell activation levels that are 3 times greater than lmg/ml of free ovalbumin (the UN. absorbance resulting from activation with 1 mg/ml of free ovalbumin was only 0.037 versus 0.1106 for activation with 1 ⁇ g/mL of ovalbumin encapsulated in the microgels).
• the acid degradable microgels are capable of delivering protein antigens into APCs for Class I antigen presentation. Higher protein loading was shown to lead to an increase in antigen presentation.
• the absorbance taken for 0.1 mg particle /mL of particles, made with the biscarylamide triglyme acetal crosslinker of Example 4, having a protein loading capacity of 22 ⁇ g protein/mg of particle is close to the same absorbance for 0.5 mg particle/mL of particles, made with the same but greater percentage of crosslinker, and having a 9.5 ⁇ g/mg loading capacity. There may be a point where there is a maximum level of antigen presentation as shown by the same absorbance of about 0.33 with 0.5 mg/mL of particles having 22.0 and 62.6 ⁇ g protein/mg of particle loading capacity.
• Toxicity of Microgels made with Bisacrylamide Acetal Crosslinker The toxicity of bioactive material loaded microgels was measured with the yellow tetrazolium salt, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), assay using RAW 309.CR1 macrophage cells (ATCC No. TIB-69, American Type Culture Collection, Manassas, VA). The cells are incubated with microgel particles in DMEM media with 10% F.B.S. The microgels were aspirated from the cells, and they were then washed several times with PBS and allowed to grow for 24-48 hours.
• MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide
• the cell viability is determined by measuring the absorbance of the reduced MTT reagent using the protocol described in Freshney et al. (Freshney, I. R. (1994) Culture of animal cells, Wiley-Liss, Inc, New York, NY) as compared to a control.
• MTT yellow
• MTT is reduced metabolically by healthy cells in part by the action of dehydrogenase enzymes in mitochondria, to generate purple formazan crystals, which are solubilized by the addition of a detergent and the absorbance is measured at 570 nm.
• the measurement of the ability of cells to reduce the MTT reagent metabolically is a measurement of the health of the cell population.
• RAW 309.CR1 macrophage cells were split at 5 xlO 4 cells per well in a 96 well plate and allowed to grow overnight. The cells were then incubated with the microgel particles (1.6 % crosslinked, sample A from Table 1 in Example 11) with variable amounts of loaded ovalbumin for 24 hours in DMEM media with 10% F.B.S. The microgels were aspirated from the cells, and they were then washed several times with PBS and allowed to grow for another 24 hours.
• the cell viability was determined by measuring the absorbance of the reduced MTT reagent.
• the MTT assay was performed using 0.5, 1, 2.5 and 5 mg particles/mL serum in each well with a microgel loading of 10 micrograms protein mg microgel particle. After 24 hours, there were almost 100% viable cells remaining in the 0.5mg particles/mL, 90% viable cells remaining in the 1 mg protein/mg particles, about 80% viable cells remaining in the 2.5 mg particles/mL, and about 80% viable cells remaining in the 5 mg particles/mL. Thus, it can be found that the microgels of the invention are not toxic to mammalian cells because more than 50% of the cells remain viable.
• RAW 309.CR1 macrophage cells were split at 5 xlO 4 cells per well in a 96 well plate and allowed to grow overnight. The cells were then incubated with the microgel particles made with the bisacrylamide tetraglyme acetal crosslinker (1.6 % crosslinked, 9:1 ratio, sample A and 12.8 % crosslinked, 1:1 ratio sample C from Table 1 in Example 11) with variable amounts of loaded oavalbumin for 20 hours in DMEM media with 10% F.B.S. The particles were aspirated from the cells, and they were then washed several times with PBS and allowed to grow for another 24 hours.
• the MTT assay was performed using 0.5, 1, 2.5 and 5 mg protein/mL serum in each well with a particle loading of 10 micrograms protein/mg microparticle After 24 hours, cells incubated with the particles having 12.8% crosslinker showed 80% viable cells remaining after exposure to 0.1 mg/mL of particles, 74% viable cells remaining in 0.5mg/mL particles, 62% viable cells remaining in 1.0 mg/mL particles, 63% viable cells remaining in 2.5 mg/mL particles, and 47% viable cells remaining in 5 mg/mL particles.
• cells incubated with the particles having 12.8% crosslinker showed 70% viable cells remaining in 0.5mg particles/mL, 63% viable cells remaining in 0.5mg particles/mL, 62% viable cells remaining in the 1 mg protein/mg particles, about 62% viable cells remaining in the 1 and 2.5 mg protein/mL particles, and about 52% viable cells remaining in the 5 mg protein/mL particles.
• Microgel particles were made with acrylamide and bisacrylamide triglyme acetal crosslinker 606 encapsulating fluorescently labeled dextran (because it easier to label and observe than fluorescence-labeled DNA) and fed to macrophage cells.
• a bisacrylamide methylene nondegradable crosslinker is used, the fluorescence is more localized showing that when nondegradable microgels have been taken up by the cells, they remain sequestered in the lysosome without a mechanism of release.
• the acid degradable bisacrylamide triglyme acetal crosslinker is used to make the microgels, the fluorescence is more diffuse within the cytoplasm of cells, which is indicative of cytoplasmic release of the microgel contents.
• dextran microgel particles were made according to Example 9 using the crosslinker of Example 8. After degradation of the microgels, (activated) dextran of 10,000 MW is easily secreted from the body and should exhibit no toxicity problems because it is a sugar. The bioactive material 40 is released along with the linker group upon hydrolysis.
• protein loading must be about 10 ⁇ g protein mg particle before the antigen presentation assay of
• Example 17 shows absorbance of 0.25 which is the target absorbance at which there is 100% T- cell activation. Dextran microgel particles also enhance antigen presentation versus free protein but about 1/3 as efficient as using acrylamide as the polymerizing group. One other concern with dextran microgels is the dispersability of the particles in solution because dextran may not be sufficiently hydrophilic. EXAMPLE 17
• Synthesis of gel particles encapsulating plasmid DNA was as follows. Plasmid DNA (pSV- ⁇ -gal vector, 6820 bp, Amp resistant) is added directly to the aqueous phase of an inverse microemulsion. The procedure is directly analogous to that for the protein loaded microgel particles made in Example 7. The organic phase consisted of hexane with 3% of the surfactants: 3/1 SPAN 80/TWEEN 80. Acrylamide monomer and the bisacrylamide triglyme acetal crosslinker (in a 4/1 mass ratio), potassium persulfate, and 250 ng plasmid DNA were dissolved in 300 mM PBS, pH 8.0.
• the two phases are combined and sonicated for 30 sec, at which point, TMEDA (tetramethylethylenediamine) was added, and the polymerization allowed to proceed for 10 min.
• TMEDA tetramethylethylenediamine
• the microgels were collected by centrifugation (10 min x 3000 RPM) and washed once with hexane and twice with acetone, then dried under vacuum overnight.
• the microgels were suspended in pH 7.4 buffer (300 mM PBS) to a concentration of 5 mg/mL. They were then collected by centrifugation, and the supernatant was removed by pipet. This step serves to remove any DNA that is adsorbed to the surface of the microgels but is not actually incorporated inside.
• the microgels were then taken up in pH 5.0 buffer (300 mM acetic acid) and incubated at 37 °C overnight for 12-18 hours. The acidic pH of the buffer cleaves the acetal linkage in the crosslinker moiety, producing linear polymer chains and free DNA.
• the plasmid DNA was then quantified by fluorescence using PICOGREENTM intercalation (Molecular Probes, Eugene, OR), a fluorescent dye that binds only double stranded DNA.
• Table 4 shows the estimated loading efficiency of the microgels made with the bisacrylamide triglyme acetal crosslinker. The highest amount loaded was 4 ⁇ g DNA mg of linear polymerizing group, however, the maximum amount of DNA that can be loaded into a sphere has not yet been reached.
• plasmid DNA There are three forms of plasmid DNA, which is normally circular: 1) supercoiled, where the circular plasmid is further coiled; 2) open circular, where the plasmid has become untwisted but is still circular; and 3) linear. Open circular and supercoiled plasmid DNA both undergo transcription. When digested with Hind HI and Xmn I restriction enzymes, the plasmid is cut twice into portions of 2263 bp and 4557 bp. The control was the DNA before transformation with DH5 ⁇ E. coli,.
• the DNA was subjected to a restriction digest with Hind HI and Xmn I and then analyzed by gel electrophoresis (0.7% agarose, 50V for 150 minutes) and post stained with ethidium bromide (gel not shown).
• the lanes of control and released DNA subject to the double digest looked identical with some linear single cut plasmid still present.
• the DNA Prior to encapsulation, the DNA is mostly supercoiled (lower band at 4361 bp) with some open circular. After sonication, vacuum drying, and exposure to acidic solution for 18 hours, the DNA is mostly open circular with some linear and supercoiled structures. Supercoiled and open circular plasmid DNA are still able to undergo transcription in cells but linear DNA cannot.
• DNA When DNA is isolated from bacteria and loaded into the microgels, the DNA was mostly supercoiled. When isolated from the microgels s, the DNA was mostly open circular, with some linear and some supercoiled structure. The DNA at this point had been through three major reaction conditions: sonication, radical polymerization, and acid exposure (pH 5, 37° C, 18 h). It is quite remarkable that that the DNA remained intact because sonication of naked DNA is known to shear and break it into linear strands or fragments. DNA is known to withstand acidic conditions, but it is rare to observe any kinetics after exposure to an acidic pH for such an extended period of time. The restriction digest (single and double cut) serves to give a footprint, demonstrating that the DNA that was encapsulated and went in to the polymerization had the same footprint as the DNA that was recovered.
• microgel Particle Toxicity of DNA Encapsulated Microgel Particle.
• the microgels were tested for toxicity using the MTT assay of Example 14.
• RAW 309.CR1 macrophage cells were split at 5 xlO 4 cells per well in a 96 well plate and allowed to grow overnight.
• the cells were then incubated with the microgel particles (1.6 % crosslinked, sample A from Table 1 in Example 11) with variable amounts of loaded DNA for 16 hours in DMEM media with 10% F.B.S.
• the particles were aspirated from the cells, and they were then washed several times with PBS and allowed to grow for another 48 hours.
• the cell viability was determined by measuring the absorbance of the reduced
• MTT reagent The MTT assay was performed using 5 mg microgels /mL serum in each well with a particle loading of 0, 1, 2 and 4 ⁇ g DNA mg microgels. After 48 hours, there were 82% viable cells remaining in the empty microgels, 70% viable cells remaining in the 1 ⁇ g DNA /mg microgels, 75% viable cells remaining in the 2 ⁇ g DNA /mg microgels, and 65% viable cells remaining in the 4 ⁇ g DNA mg microgels. The concentration tested (5 mg microgels / mL serum) is quite high for most applications, so a toxicity of 80% viability is permissible. It can be concluded that neither the polyacrylamide microgels nor the DNA is toxic to the macrophage cells because at least 50% viable cells are left.
• microgel particles were also tested for DNA release.
• the microgel particles were suspended in either pH 7.4 or pH 5.0 buffer.
• the amount of DNA released into the supernatant was quantified by fluorescence using PICOGREENTM (Molecular Probes, Eugene, OR).
• PICOGREENTM Molecular Probes, Eugene, OR.
• pH 7.4 there is an initial burst, as is also seen with the protein loaded microgel particles in Example 12. This is most likely due to DNA that is adsorbed onto the surface.
• pH 5 all of the DNA is readily released within two hours.
• the microgels are visually degraded after 30 min and appear as a gel in this assay after hydrolysis.
• the plasmid DNA is physically entrapped within the microgel particles made with the bisacrylamide triglyme acetal crosslinker, the DNA is protected from otherwise being degraded in the serum. DNase enzymes readily chew up naked DNA in serum, however the encapsulated DNA showed good stability.
• the microgels were incubated in serum (90% DMEM, 10% FBS) for a set period of time of 24 hours. The microgels were then collected, and the serum supernatant removed.
• the plasmid DNA was isolated from the microgel particles by placing in acetic acid for 6 hours at pH 5.0. Following hydrolysis of the microgels, the DNA released was quantified using PICOGREENTM.
• Kidney 293T cells (ATCC, Manassas, VA) is a kidney cell line, relatively easy to transfect.
• the 293T cells are treated with DNA and Lipofectamine 2000 (Promega, Madison, WI), a cationic lipid, a known transfection agent. If the DNA that is recovered is intact, the cells should produce •-galactosidase upon transfection. After 24h, the cells were lysed and a galacto-ortho-nitrophenol substrate was added. If '-galactosidase is present, the acetal bond in galacto-ortho-nitrophenol is cleaved and the released phenolate turns purple and absorbs at 570 nm.
• plasmid DNA alone to the cells causes no transfection and no •- galactosidase activity is detected.
• Cells that were transfected by the control plasmid DNA showed an absorbance of 0.55, 0.6 and 1.0 at DNA concentrations of 0.25 ng, 0.50 ng and 1.0 ng.
• Cells transfected with open circular DNA isolated from the microgel particles also shows •-galactosidase activity and had absorbances of 0.4 and 0.55 at 0.25 ng and 0.50 ng respectively.
• Interleukin-6 was detected by an ELISA assay.
• RAW 264.7 macrophages were incubated with either microgels encapsulating plasmid DNA or naked plasmid overnight.
• the supernatant was analyzed for IL-6 production using an ELISA kit (Pierce Biotechnology, Rockford, IL).
• the IL-6 enzyme levels were detected for the following amounts of DNA added to each well: untreated (control), 1 ⁇ g of DNA encoding ⁇ -galactosidase, 1 ⁇ g plasmid DNA + lipofectamine, unloaded microgels, 0.1 ⁇ g plasmid DNA in microgels, 0.2 ⁇ g plasmid DNA in microgels, and 0.4 ⁇ g plasmid DNA in microgels.
• Untreated cells as well as those incubated with plasmid DNA show a low level of about 300-400 pg/mL of secreted IL-6.
• the IL-6 level is increased to about 2000 pg/mL of IL-6.
• Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, CA) is a commercially available transfection agent that forms micelles around the DNA, protecting it from nucleases in the serum and facilitating cellular delivery. Therefore, this shows that naked DNA requires a transfection agent, such as Lipofectamine, in order to produce an immune response.
• Unloaded microgels had no IL-6 response, meaning that microgels alone do not induce an immune response.
• Microgels with DNA did induce IL-6 secretion. When as little as 0.1 ⁇ g of DNA is delivered per well, an immune response of about 350 pg/ml of H--g is observed. When 0.2 ⁇ g of plasmid DNA is delivered in microgels, there is about 1700 pg/mL of IL-6 detected. This is equivalent to a 30-fold increase in IL-6 production when compared to naked DNA alone, for which 1 ug is delivered per well..
• Macrophages also release other mediators including prostraglandins, oxygen radicals, peroxides, NO, etc. Comparing these same samples again, there was a 70-fold enhancement in NO activity when the DNA is encapsulated in the microgels. Nitric oxide is detected by the Griess assay by measuring NO 2 " .
• RAW 309.1 cells were split onto a 96 well plate at 4 x 10 5 cells per well the night before the experiment and grown in 10% serum containing DMEM medium. The medium was then removed and the appropriate microparticle sample was added, and the cells were grown in serum containing media for 16 hours.
• the media was then aspirated off and the cells were stimulated with 10 units/ml of gamma interferon and lO ⁇ g/ml of LPS for 8 hours in serum containing media (to stimulate NO 2 production).
• the medium was then isolated and the concentration of NO was measured by mixing the supernatant with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylene-diamine hydrochloride, and 5% phosphoric acid).
• the absorbance at 540nm was measured after 10 minutes at room temperature.
• microgel particles are made with the crosslinkers of Example 4 or 5.
• the dendritic cells are pulsed at an immature stage and then cultured with ovalbumin (OVA) transgenic CD4 and CD8 T-cells for several days.
• OVA ovalbumin
• the following groups can be tested: microgels entrapping OVA, microgels entrapping OVA +TNF, microgels entrapping protein control, microparticles entrapping protein control +TNF, OVA alone, OVA+TNF, and peptide + TNF.
• the amount of OVA used should be about 50-100 ⁇ g /ml, which means that approximately 250-500 ⁇ g of protein total or less should be entrapped within the microgels.
• a similar amount of microgels that do not contain OVA or that contains a different protein is required for a control.
• the microparticles are injected into the food pad of CD4 or CD8 transgenic mice to show that microgels can activate cytotoxic T lymphocytes in vivo. More preferably, delivery is by injection of 50 ⁇ l of resuspended particle using a 25 gauge syringe in the flanks of these transgenic mice. At least 50 ⁇ g of OV A/mouse should suffice per injection with at least 3 mice per group injected. Also 150 ⁇ g of microgels with OVA and a similar amount of mirogels used for control are injected. The lymph nodes are isolated 7 days after the injection and analyzed for antigen specific T cell priming.

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